Cloning and Expression Analysis of BjuEAR1-1 in Tuber Mustard

doi:10.7668/hbnxb.20192689

Abstract

Abstract:

During the growth and development stages,tuber mustard is always affected by abiotic stresses.As a plant stress hormone,ABA plays an important role in regulating plant response to abiotic stress.BjuEAR1-1 was a homologue of negative regulator EAR1 in ABA signaling pathway,and its mutant ear1 exhibited an ABA sensitive phenotype.Therefore,to explore the biological function of BjuEAR1-1 will provide a direction for the regulation mechanism of resistance to adverse environment stresses,and also provide an important theoretical basis for the effective use of gene resources to cultivate new varieties of BjuEAR1-1 with resistance to adverse stresses and stable yield.The BjuEAR1-1 coding sequence was cloned by PCR method and the plant overexpression vector 35S∷PTF101-GFP-BjuEAR1-1 was constructed by enzyme digestion and ligation method.The subcellular localization of BjuEAR1-1 was analyzed by transient transformation technology of tobacco leaves.The cis-element of BjuEAR1-1 gene promoter was analyzed,and the gene expression patterns of BjuEAR1-1 under different abiotic stresses and different tissues and organs were detected by fluorescence quantitative PCR.The result of subcellular localization assay showed that BjuEAR1-1 was localized in the nucleus and cytoplasm of tobacco.BjuEAR1-1 was expressed in root,stem,leaf,flower,seed pod and swollen stem of tuber mustard,with the highest expression in root.The expression level of BjuEAR1-1 was significantly induced under low temperature and ABA stresses,and there was no significant difference under salt stress. BjuEAR1-1 was localized in the nucleus and cytoplasm and regulated the response of tuber mustard to low temperature and ABA stresses.

Key words: Tuber mustard, Abiotic stress, BjuEAR1-1, Gene clone, Subcellular localization, Gene expression

CHENG Chunhong, YOU Jingfan, CAI Zhaoming, YE Chunhong, CHEN Li. Cloning and Expression Analysis of BjuEAR1-1 in Tuber Mustard[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(3): 37-43. doi: 10.7668/hbnxb.20192689.

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http://www.hbnxb.net/EN/Y2022/V37/I3/37

Figures/Tables 6

Fig.1 Gene cloning and T vector ligation of BjuEAR1-1 in tuber mustard A.Amplification result of CDS sequence of BjuEAR1-1 in tuber mustard:M.DL2000 DNA Marker;1-2.Amplification of CDS sequence of BjuEAR1-1.B.Screening of BjuEAR1-1-T transformant plate.C.Identification of BjuEAR1-1-T colony by PCR:M.DL2000 DNA Marker;1-6.Identification of BjuEAR1-1-T colony by PCR.

Fig.2 Vector construction of 35S∷PTF101-GFP-BjuEAR1-1 A.Detection of target fragments after BjuEAR1-1-T and 35S∷PTF101-GFP double enzyme digestion:M.DL2000 DNA Marker;1-2.Target fragment recovery after BjuEAR1-1-T double digestion;3.Target fragment recovery after 35S∷PTF101-GFP double enzyme digestion.B.Detection of 35S∷PTF101-GFP-BjuEAR1-1 colony by PCR:M.DL2000 DNA Marker;1-6.Detection of colony by PCR.C.Enzyme digestion identification of 35S∷PTF101-GFP-BjuEAR1-1:M.DL2000 DNA Marker;1-4.35S∷PTF101-GFP-BjuEAR1-1 plasmid digestion identification.

Fig.3 Subcellular localization analysis of BjuEAR1-1

Fig.4 The cis-acting elements analysis of BjuEAR1-1 promoter

Fig.5 Analysis of temporal and spatial expression patterns of BjuEAR1-1 in different tissues and organs The different normal letters indicate significant difference between treatments at the 0.05 level.The same as Fig.6.

Fig.6 Expression analysis of BjuEAR1-1 under different stresses treatment

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